The causative agent for Acquired Immunodeficiency Syndrome (AIDS) is the Human Immunodeficiency Virus (Gallo et al. (1993) Science 220:865-867; Barre-Sinoussi et al. (1983) Science 220:868-870; Shaw et al. (1984) Science 226:1165-1171). Like all known retroviruses, HIV must reverse transcribe its RNA genome and integrate the double-stranded DNA copy into the host genome (for review; Wong-Staal (1990) In Virology 2nd Ed. (B. N. Fields et al. eds) Raven Press, N.Y. pp. 1529-1543; Cann and Chen (1990) In Virology 2nd Ed. (B. N. Fields et al. eds) Raven Press, New York pp. 1501-1527; Vaishnav and Wong-Staal (1991) Ann. Rev. Biochem. 60:577-630). The viral component that is essential for formation of a provirus is the integrase protein (Schwartzberg et al. (1984) Cell 37:1043-1052; Donebower and Varmus (1984) Proc. Natl. Acad. Sci. U.S.A. 81:6461-6465; Craigie et al. (1990) Cell 62:829-837). Integrase, in vitro, has been shown to be necessary and sufficient for processing of the double-stranded viral DNA (processing or donor cut; Katzman et al. (1989) J. Virol. 63:5319-5327; Sherman and Fyfe (1990) Proc. Natl. Acad Sci. U.S.A. 87:5119-5123), cleaving recipient DNA and ligating processed DNA to it (joining or strand transfer; Grandgenett et al. (1986) J. Virol. 58:970-974; Bushman and Craigie (1991) Proc. Natl. Acad. Sci. U.S.A. 88:1339-1343) and for an event that is yet to be demonstrated in vivo, resolution of integrated DNA to component parts (disintegration; Chow et al. (1992) Science 255:723-726; Chow and Brown (1994) J. Virol. 68:3896-3907). The protein has been divided into three structural domains. The N-terminal domain is highly conserved among retroviral integrases and encodes a Zn++ finger-like DNA binding motif. While the C-terminal domain is variable but consistently basic, with a net charge of about +11. Integrase associates with the double-stranded HIV DNA to form a pre-integration complex which is transported into the nucleus of infected cells (Bowerman et al. (1989) Genes and Development 3:469-478; reviews: Goff (1992) Ann. Rev. Genet. 26:527-544; Whitcomb and Hughes (1992) Ann. Rev. Cell Biol. 8:275-306). It has been suggested that integrase encodes a nuclear localization signal in the C-terminal domain. Mutational analysis of the different domains and the results from complementation tests suggest that integrase functions as a multimer rather than a monomer (Jones et al. (1992) J. Biol. Chem. 267:16037-16040; Engelman et al. (1993) EMBO J. 12:3269-3275; Leavitt et al. (1993) J. Biol. Chem. 268:2113-2119). This may explain how this enzyme is able to cleave different DNA sequences and remain associated with multiple ends of DNA at the same time.
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, entitled Systematic Evolution of Ligands by Exponential Enrichment, now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled Nucleic Acid Ligands, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,270,163 (see also PCT/US91/04078), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled Method for Selecting Nucleic Acids on the Basis of Structure, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled Photoselection of Nucleic Acid Ligands describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed "counter-SELEX." U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled High Affinity Nucleic Acid Ligands Containing Modified Nucleotides, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2'-amino modifications. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'--NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). Each of these applications is specifically incorporated herein by reference.
A SELEX-like process was used by Barrel et al. to identify the important structural features of the vira RNA element bound by the Rev protein of HIV-1. (Barrel et al. (1991) Cell 67:529-536.) In one of three rounds of selection performed, wild-type RNA was included in the reaction mixture to compete with the pool RNA for binding to the target protein.
The development of high affinity ligands capable of inhibiting HIV integrase would be useful in the treatment of Human Immunodeficiency Virus. Herein described are high affinity RNA ligand inhibitors of HIV integrase.